The present invention relates to a reflected fluorescence microscope, and more particularly to a reflected fluorescence microscope employing the laser trap method or the Caged reagent release method used as a means for cellular operation in the cell physiological field.
The reflected fluorescence observation using a reflected fluorescence microscope is now a method widely and generally employed as a method of morphologic observation of specific substances inside cells in the biological field.
Recently, the laser trap method or the Caged reagent release method has been widely employed as an operating intracellular substances operating method using a reflected illumination means of a microscope, particularly, in the cell physiological field.
The laser trap method (light pin set method), which is a method of applying laser light to an arbitrary substance in a sample and optically capturing the substance, is employed as a means for measuring energy when protein in cells moves. Infrared laser light is used for the applying means. On the other hand, the Caged reagent release method is a method of bonding a Caged reagent with molecules having physiological activity, administrating it into cells in a state where activity is restricted, and releasing the bonding by applying light thereto so as to recover the activity of the cells. Ultraviolet light is used for the applying means.
As described above, a reflected fluorescence microscope employing the laser trap method or the Caged reagent release method needs various light sources for cellular operation other than the general reflected fluorescence illumination.
In a conventionally used general reflected fluorescence microscope, as shown in FIG. 1, an excitation light source 104 for fluorescence observation is provided for a first dichroic mirror DM1 provided together with a barrier filter BA in an observation optical system light path 103 connecting an object lens 101 and an eyepiece 102, a second dichroic mirror DM2 is provided between the fist dichroic mirror DM1 and the excitation light source 104, a band pass filter BP is provided between the second dichroic mirror DM2 and the excitation light source 104, and a light source 105 for sample operation is provided in a light path which is orthogonal at the second dichroic mirror DM2.
Further, Jpn. Pat. Appln. KOKAI Publication No. 8-234110 discloses a reflected fluorescence microscope, as shown in FIG. 2, wherein a first dichroic mirror DM1 for introducing excitation light from an excitation light source 108 for fluorescence observation into an observation optical system light path 107, and a second dichroic mirror DM2 for introducing laser light of the laser trap from a laser light source 109 thereto are provided at two stages.
In the prior art shown in FIG. 1, however, not only the band pass filter BP, but also the first dichroic mirror DM1 need to be exchanged if executing the fluorescence observation by switching the excitation wavelength of the excitation light source 104 is considered. At this time, the first dichroic mirror DM1 and the band pass filter BP, which are provided separately from one another, must be exchanged separately, and the work for this exchange is complicated and troublesome.
On the other hand, in the prior art in FIG. 2 disclosed in Jpn. Pat. Appln. KOKAI Publication No. 8-234110, in a case where the first dichroic mirror DM1 is provided more closely to the sample side than the second dichroic mirror DM2, if, for example, the laser wavelength range of the laser light source 109 is changed to the ultraviolet range, which is to be used for the Caged reagent release, the first dichroic mirror DM1 and the barrier filter BA must have a characteristic of transmitting the laser wavelength from the laser light source 109 in order to certainly introduce the laser light reflected at the second dichroic mirror DM2 into the sample side. At every introduction of the laser light, a troublesome work such as exchange of the first dichroic mirror DM1 and the barrier filter BA to optimal ones need to be carried out and, therefore, there is little realizability of this system. Actually, the laser light source 109 cannot be applied to the object of use in the range other than the infrared range for the laser trap and can be hardly used for a general purpose. Further, since the second dichroic mirror DM2 is fixed in the observation optical system light path 107, no problems occur in a general fluorescence observation, but the fixation is a reason for loss of light amount in the tiny fluorescence observation such as measurement of light at the one-molecule level and is not therefore preferable.